Pediococcus acidilactici TCI550, and use thereof for neurological health care and / or cognition enhancement

Abstract

Pediococcus acidilactici, which is Pediococcus acidilactici TCI550 having accession number DSM34536. In addition, the Pediococcus acidilactici TCI550 and / or a metabolite thereof is suitable for use in the preparation of compositions for neurological health care and / or cognitive enhancement.

Description

Pediococcus acidilactici TCI550 and uses thereof for neuroprotection and / or cognitive enhancement TECHNICAL FIELD

[0001] The present disclosure relates to a Pediococcus acidilactici and uses thereof, in particular to a Pediococcus acidilactici TCI550 and / or metabolites thereof, which can be used for preparing a neuroprotective and / or cognitive enhancing composition. BACKGROUND

[0002] Probiotics generally refer to ingested microorganisms that have positive effects on the host (or recipient, such as animals or humans) after ingestion. The name is derived from the Greek word "for life" (beneficial to life), also known as "probiotic health bacteria". Moreover, probiotics mainly refer to lactic acid bacteria and some yeast bacteria.

[0003] Generally speaking, "lactic acid bacteria" refers to a group of bacteria that can utilize carbohydrates for fermentation to produce a large amount of lactic acid. Among the many species of lactic acid bacteria, some strains of lactic acid bacteria have been found to be beneficial to the health of the recipient, and therefore these strains are considered probiotics. In other words, most probiotics are lactic acid bacteria, but only a few strains of lactic acid bacteria that have been proven to be beneficial to the health of the recipient can be called probiotics.

[0004] For example, common lactic acid bacteria species that can be used as probiotics include Enterococcus, Lactobacillus, Bifidobacterium, Bacillus, etc., and yeast species that can be used as probiotics include Saccharomyces.

[0005] Lactobacillus is a group of gram-positive bacilli with fermentative ability, facultative anaerobic, and no spore production. Lactobacillus is named for its ability to ferment carbohydrates into lactic acid, and can be used to make liquid yogurt, solid cheese, German sauerkraut, beer, wine, pickles, pickled foods and other fermented foods. SUMMARY

[0006] Therefore, the present disclosure provides a Pediococcus acidilactici TCI550 and / or metabolites thereof, which has neuroprotective and / or cognitive enhancing functions.

[0007] In some embodiments, a Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the deposit number DSM34536.

[0008] In some embodiments, use of a Pediococcus acidilactici and / or metabolites thereof for the manufacture of a composition for neurohealth, wherein the Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the deposit number DSM34536.

[0009] In some embodiments, the neurohealth is improving brain plasticity, calming the nervous system, or a combination thereof.

[0010] In some embodiments, the aforementioned composition for neurohealth has the ability to reduce the overall activity of sympathetic nervous cells.

[0011] In some embodiments, use of a Pediococcus acidilactici and / or metabolites thereof for the manufacture of a composition for improving cognition, wherein the Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the deposit number DSM34536.

[0012] In some embodiments, the aforementioned composition for improving cognition has the ability to improve reactivity, focus, memory, or a combination thereof.

[0013] In some embodiments, the aforementioned composition has the ability to promote the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof.

[0014] In some embodiments, the aforementioned serotonin production-related gene is a Tryptophan hydroxylase (TPH) gene, a Dopa decarboxylase (DDC) gene, or a combination thereof.

[0015] In some embodiments, the aforementioned melatonin synthesis-related gene is an Acetylserotonin O-methyltransferase (ASMT) gene, an Arylalkylamine acetyltransferase (AANAT) gene, or a combination thereof.

[0016] In some embodiments, the aforementioned anti-depression-related gene is a Sirtuin 1 (SIRT1) gene, a Brain-derived neurotrophic factor gene, or a combination thereof.

[0017] In some embodiments, the aforementioned composition has the ability to promote the secretion of γ-aminobutyric acid (GABA) by neural cells.

[0018] In some embodiments, the Pediococcus acidilactici TCI550 has the ability to produce GABA and to increase the GABA content in blood.

[0019] In some embodiments, the Pediococcus acidilactici TCI550 has the ability to increase the Brain-Derived Neurotrophic Factor (BDNF) content in blood.

[0020] In some embodiments, the Pediococcus acidilactici TCI550 is used in an amount of 50 mg per day.

[0021] In some embodiments, the Pediococcus acidilactici TCI550 and / or its metabolites are used in the preparation of a composition. The composition has a neuroprotective and / or cognitive enhancing effect. In some embodiments, the composition has at least one of the following abilities: producing GABA, increasing the GABA content in blood, and increasing the Brain-Derived Neurotrophic Factor (BDNF) content in blood. In some embodiments, the composition has at least one of the following abilities: increasing the expression of a serotonin production related gene, a melatonin synthesis related gene, and an anti-depression related gene in a neural cell. The serotonin production related gene can be, but is not limited to, a tryptophan hydroxylase gene, a dopamine decarboxylase gene, or a combination thereof; the melatonin synthesis related gene can be, but is not limited to, an acetylserotonin O-methyltransferase (ASMT) gene, an aralkylamine acetyltransferase (AANAT) gene, or a combination thereof; the anti-depression related gene can be, but is not limited to, a Sirtuin 1 (SIRT1) gene, a Brain-Derived Neurotrophic Factor (BDNF) gene, or a combination thereof. In some embodiments, the composition has a neuroprotective effect by at least one of the following abilities: reducing the overall activity of sympathetic neural cells, increasing the brain plasticity, and calming the neural system. In some embodiments, the composition has a cognitive enhancing effect by at least one of the following abilities: increasing the reaction, concentration, and memory. BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a graph showing the GABA detection data of various Lactobacillus and Pediococcus acidilactici;

[0023] FIG. 2 is a graph showing the GABA detection data of neural progenitor cells;

[0024] FIG. 3 is a graph showing the BDNF gene expression data of neural progenitor cells;

[0025] FIG. 4 is a graph showing the analysis results of serotonin production related genes, melatonin synthesis related genes, and anti-depression related genes.

[0026] FIG. 5 is a graph showing the average gamma-aminobutyric acid (GABA) content in blood of the subjects at week 0, week 2, and week 4;

[0027] FIG. 6 is a graph showing the average brain-derived neurotrophic factor (BDNF) content in blood of the subjects at week 0, week 2, and week 4;

[0028] FIG. 7 is a graph showing the sympathetic nerve cell activity of the subjects at week 0 and week 4;

[0029] FIG. 8 is a graph showing the results of the sleep disorder severity assessment of the subjects at week 0 and week 4;

[0030] FIG. 9 is a graph showing the results of the sleep quality severity assessment of the subjects at week 0 and week 4;

[0031] FIG. 10 is a graph showing the results of the cognitive dysfunction score of the subjects at week 0 and week 4;

[0032] FIG. 11 is a graph showing the results of the cognitive function severity assessment of the subjects at week 0 and week 4;

[0033] FIG. 12 is a graph showing the results of the somatosensory questionnaire analysis of the subjects at week 4 for improving the frequency of waking up at midnight; and

[0034] FIG. 13 is a graph showing the results of the somatosensory questionnaire analysis of the subjects at week 4 for improving concentration.

[0035] Preservation of biological material

[0036] Pediococcus acidilactici TCI550, deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ); date of deposit: 17 February 2023; accession number: DSM34536. DETAILED DESCRIPTION

[0037] Pediococcus acidilactici TCI550 is a strain of the genus Pediococcus of the family of Lactobacillaceae of the order of Lactobacillales. Pediococcus acidilactici TCI550 is in the form of a sphere and is usually found in pairs or tetrad. Pediococcus acidilactici TCI550 is a gram-positive bacterium, can grow and multiply in an anaerobic environment, and is an anaerobic bacterium. In some embodiments, Pediococcus acidilactici TCI550 can be directly isolated from the human intestinal tract.

[0038] P. acidilactici TCI550 grows at temperatures between 25°C and 45°C, preferably between 30°C and 37°C.

[0039] P. acidilactici TCI550 is acid tolerant and can survive in lower pH environments (e.g. in yogurt and pickled foods). In this regard, P. acidilactici TCI550 can survive in environments with a pH between 4 and 7. In some embodiments, P. acidilactici TCI550 can generally tolerate acidic environments with a pH of 4.0 or even lower.

[0040] P. acidilactici TCI550 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) on February 17, 2023 under the accession number DSM34536.

[0041] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the ability to increase the content of gamma-aminobutyric acid (GABA). GABA can be secreted by P. acidilactici TCI550. In addition, GABA can also be induced by P. acidilactici TCI550 and / or its metabolites to be secreted by other cells. In some embodiments, P. acidilactici TCI550 can have the ability to increase the content of GABA in the blood of a recipient. For example, when a recipient consumes P. acidilactici TCI550, P. acidilactici TCI550 colonized in the recipient's body (e.g. the intestinal tract) produces GABA, and P. acidilactici TCI550 also promotes nerve cells (e.g. neural progenitor cells) to secrete GABA, thereby increasing the content of GABA in the blood of the recipient. Moreover, when GABA acts on nerve cells in the brain, it binds to GABA receptors to open chloride channels on the cell membrane and trigger inhibitory action potentials to calm the nerves. Therefore, GABA helps to relax the body and mind and aids sleep.

[0042] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the ability to increase the expression of brain-derived neurotrophic factor gene (hereinafter referred to as BDNF gene) of neural cells, and to promote the content of brain-derived neurotrophic factor (BDNF) in the blood of the recipient. For example, when the recipient takes P. acidilactici TCI550, the P. acidilactici TCI550 and its metabolites colonized in the body of the recipient (such as the intestinal tract) promote the expression of BDNF gene of neural cells (such as neural progenitor cells), and secrete brain-derived neurotrophic factor, so as to increase the content of brain-derived neurotrophic factor in the blood of the recipient. The increase of brain-derived neurotrophic factor helps neural plasticity and brain strength, so that P. acidilactici TCI550 and / or its metabolites help the neural plasticity of the recipient.

[0043] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the function of neuroprotection. Among them, neuroprotection refers to the functions of improving brain plasticity, calming nerves, etc. For example, after the recipient takes P. acidilactici TCI550, the brain plasticity of the recipient can be improved, and the nerves of the recipient can be calmed.

[0044] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the ability to reduce the overall activity of sympathetic nerve cells of the recipient. For example, after the recipient takes P. acidilactici TCI550 and / or its metabolites, the overall activity of sympathetic nerve cells of the recipient decreases. When the autonomic nervous system of the recipient is out of balance, the sympathetic nerves will be in a state of hyperactivity during sleep, so that the body and brain cannot rest properly. Therefore, when the overall activity of the sympathetic nerves of the recipient decreases after taking P. acidilactici TCI550 and / or its metabolites, the recipient's tense and excited mood can be reduced, and the recipient can fall asleep.

[0045] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the function of improving cognition. Specifically, P. acidilactici TCI550 and / or its metabolites can improve the reaction, concentration, memory and other cognitive indicators of the recipient. For example, after the recipient takes P. acidilactici TCI550 and / or its metabolites, the reaction, concentration, memory and other cognitive indicators of the recipient can be improved, and the cognitive ability of the recipient can be improved.

[0046] In some embodiments, the P. acidipiscicola TCI550 and / or its metabolites have the ability to increase the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in neural cells. The serotonin production-related gene is a tryptophan hydroxylase (TPH) gene, a dopa decarboxylase (DDC) gene, or a combination thereof; the melatonin synthesis-related gene is an acetylserotonin O-methyltransferase (ASMT) gene, an aralkylamine acetyltransferase (AANAT) gene, or a combination thereof; and the anti-depression-related gene is a sirtuin 1 (SIRT1) gene, a brain-derived neurotrophic factor (BDNF) gene, or a combination thereof.

[0047] The TPH gene encodes a tryptophan hydroxylase that catalyzes the formation of serotonin, and the tryptophan hydroxylase is believed to play an important role in mood-related diseases such as depression. The DDC gene encodes a protein that catalyzes the formation of serotonin. Serotonin is an important factor related to learning and memory.

[0048] The ASMT gene encodes a protein that catalyzes the formation of melatonin. The AANAT gene encodes a protein that converts serotonin into N-acetylserotonin.

[0049] The SIRT1 gene de-methylates histones to promote neural differentiation. Activated SIRT1 genes help reduce inflammation in the brain, help reduce depression, and can play an important role in the pathogenesis of major depression. In addition, when the expression level of the SIRT1 gene is increased and the density of neural cells is regulated to increase, it helps reduce depression. The BDNF gene, as one of the important factors in the NTRK2 signaling pathway, promotes the survival and differentiation of neuronal populations in the peripheral and central nervous systems during development, and has various effects such as anti-depression, slowing down cognitive decline, promoting the growth of new neurons and synapses, protecting existing neurons, maintaining long-term memory, and the like.

[0050] In some embodiments, the metabolite of P. acidilactici TCI550 is an active ingredient secreted by P. acidilactici TCI550 extracellularly. In some embodiments, the metabolite of P. acidilactici TCI550 can be a TCI550 filtrate obtained after centrifugation, filtration to remove bacterial cells, etc. of the P. acidilactici TCI550 culture solution after culture, and the aforementioned filtrate contains the active ingredient secreted by P. acidilactici TCI550. For example, the bacterial solution obtained after culture of the P. acidilactici TCI550 filtrate is centrifuged at 10,000xg for 10 minutes to obtain a supernatant, and the supernatant is filtered with a 0.22um filter to obtain a TCI550 filtrate, i.e., a metabolite of P. acidilactici TCI550 (including active ingredients and culture solution).

[0051] In some embodiments, P. acidilactici TCI550 and / or its metabolite can be used to prepare a composition. The composition can be used for neuroprotection or / and cognitive enhancement.

[0052] In some embodiments, the aforementioned composition has the ability to reduce the overall activity of sympathetic nerve cells, increase the expression of genes related to the production of serotonin in nerve cells (such as TPH gene, DDC gene), genes related to the synthesis of melatonin (such as ASMT gene, AANAT gene), anti-depression related genes (such as SIRT1 gene, BDNF gene), or a combination thereof, promote the secretion of γ-aminobutyric acid in nerve cells, improve reaction, concentration, memory, or a combination thereof, etc.

[0053] Among them, γ-aminobutyric acid helps to promote neural plasticity and improve cognitive ability.

[0054] Among them, brain-derived neurotrophic factor helps brain plasticity, which can regulate transport, phosphorylation-related, and can enhance synaptic strength. And brain-derived neurotrophic factor is directly related to brain learning and memory.

[0055] In some embodiments, the aforementioned composition contains a specific amount of P. acidilactici TCI550 or its metabolite. For example, the composition has a specific use amount of P. acidilactici TCI550, and the effective use amount of P. acidilactici TCI550 is 50mg / day. Specifically, assuming that one serving of the composition is administered daily and P. acidilactici TCI550 is a dry powder, the composition contains at least 50mg of P. acidilactici TCI550.

[0056] In some embodiments, the aforementioned subject is a human.

[0057] In some embodiments, the aforementioned composition can be a health product, a food product, or a food additive for non-medical purposes. In other words, the health product, the food product, or the food additive comprises P. acidipiscis TCI550 or its metabolite in a specific amount for use.

[0058] In some embodiments, the aforementioned health product, the food product, or the food additive can further comprise a food acceptable carrier that is widely used in food manufacturing technology. For example, the food acceptable carrier can comprise one or more of the following agents: a solvent, a buffer, an emulsifier, a suspending agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the ordinary skill and routine techniques of one of ordinary skill in the art.

[0059] In some embodiments, the aforementioned food acceptable carrier of the health product, the food product, or the food additive comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), and an aqueous solution containing alcohol.

[0060] In some embodiments, the food product can be, but is not limited to, beverages, fermented foods, bakery products, health foods for non-medical purposes, and dietary supplements.

[0061] In some embodiments, the composition can be in the form of, but not limited to, powder, capsule, tablet, granule, and the like.

[0062] In some embodiments, the composition can comprise only Pediococcus acidilactici TCI550. In other embodiments, the composition comprises Pediococcus acidilactici TCI550 and at least one food additive, and the food additive is at least one of the following group: soy milk powder, trehalose, indigestible maltodextrin, and sorbitol.

[0063] In some embodiments, the composition is composed of Pediococcus acidilactici TCI550, soy milk powder, trehalose, indigestible maltodextrin, and sorbitol. In some exemplary embodiments, the composition is prepared by mixing Pediococcus acidilactici TCI550 broth, soy milk powder, trehalose, indigestible maltodextrin, and sorbitol uniformly, followed by freeze-drying and powdering. For example, Pediococcus acidilactici TCI550 is incubated at 37°C for 24 hours to obtain Pediococcus acidilactici TCI550 broth, and 1.25% (w / w) of soy milk powder, 1.25% (w / w) of trehalose, 0.5% (w / w) of indigestible maltodextrin, and 0.3% (w / w) of sorbitol are added to the Pediococcus acidilactici TCI550 broth according to the weight percentage of the broth, mixed uniformly, and then dried and ground to obtain a composition containing live Pediococcus acidilactici TCI550, i.e., a bacteria powder composed of live Pediococcus acidilactici TCI550, soy milk powder, trehalose, indigestible maltodextrin, and sorbitol.

[0064] In some embodiments, the composition can be a capsule containing live Pediococcus acidilactici TCI550 bacteria powder. For example, after freeze-drying and grinding Pediococcus acidilactici TCI550 broth into live bacteria powder, an appropriate amount (e.g., 50 mg) of live bacteria powder is filled into a capsule to obtain a capsule containing live Pediococcus acidilactici TCI550 bacteria powder. In other embodiments, the composition can be a capsule containing live Pediococcus acidilactici TCI550 bacteria powder and other food additives. For example, after mixing live bacteria powder with other food additives, an appropriate amount of the mixed powder is filled into a capsule to obtain a capsule containing live Pediococcus acidilactici TCI550.

[0065] Example 1: Strain identification

[0066] An appropriate amount of each sample was taken from different source samples (as shown in Table 1) and plated on solid medium and incubated at 37°C in an anaerobic environment (i.e. the oxygen concentration in the incubation environment is less than 1 vol%) for 16 hours to form single colonies. Here, the solid medium includes 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of potassium dihydrogen phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester eighty, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0067] Next, a plurality of single colonies were picked from the solid Lactobacillus MRS medium and subjected to species identification using the 16S ribosomal DNA (16S rDNA) sequence of lactic acid bacteria. The 16S rDNA sequence of these single colonies was obtained by polymerase chain reaction (PCR), and the similarity of the 16S rDNA sequence of these single colonies to other model or published strains was determined by performing sequence alignment of these gene sequences with the 16S rDNA sequences of other Lactobacillus species using the National Center for Biotechnology Information (NCBI) website. The possible species of these single colonies were determined based on the similarity of the 16S rDNA sequence of these single colonies to other model or published strains, and the results of the species identification are shown in Table 1. Among them, the single colony of strain number TCI550 has a similarity of 99.33% to the standard strain DSM20284 of Pediococcus acidilactici (see “Aligned species” shown in Table 1), which represents that the single colony of strain number TCI550 is one of the strains of Pediococcus acidilactici, and therefore it is named as Pediococcus acidilactici TCI550.

[0068] Table 1

[0069] In addition, the Pediococcus acidilactici TCI550 was deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with the accession number DSM34536.

[0070] Example 2: Content of γ-aminobutyric acid (GABA) secreted by lactic acid bacteria

[0071] Eight lactic acid bacteria in Table 1 (i.e., Pediococcus acidilactici TCI550, Pediococcus acidilactici LH426, Lactobacillus brevis LF417, Lactobacillus brevis LF237, Lactobacillus rhamnosus LF358, Lactobacillus rhamnosus LF165, Pediococcus acidilactici LF239, and Pediococcus acidilactici LH349) were inoculated into liquid medium and cultured at 37°C in an anaerobic environment (i.e., the oxygen concentration in the culture environment was less than 1% by volume) for 24 hours to obtain bacterial liquids to be tested. At this point, the liquid medium comprises 25 g / L of yeast extract 902, 10 g / L of yeast extract GLSF, 6 g / L of potassium phosphate dibasic, 3 g / L of potassium phosphate monobasic, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0072] Next, each group of bacterial liquids was centrifuged at 5000 rpm for 15 minutes to separate the bacterial bodies and supernatants of each group of lactic acid bacteria. Then, the GABA content of each group of supernatants was measured using a GABA content detection kit (brand CLOUD-CLONE CORP; product number CEA900Ge ELISA Kit for Gamma-Aminobutyric Acid (GABA)). The results are shown in FIG. 1, in which ** indicates P<0.01, and the comparison is between Pediococcus acidilactici TCI550 and Pediococcus acidilactici LH349.

[0073] Referring to FIG. 1, the GABA content in the supernatant of Pediococcus acidilactici TCI550 was 1936.48 mg / L, the GABA content in the supernatant of Pediococcus acidilactici LH426 was 782.06 mg / L, the GABA content in the supernatant of Lactobacillus brevis LF417 was 877.41 mg / L, the GABA content in the supernatant of Lactobacillus brevis LF237 was 939.84 mg / L, the GABA content in the supernatant of Lactobacillus rhamnosus LF358 was 1044.24 mg / L, the GABA content in the supernatant of Lactobacillus rhamnosus LF165 was 1046.32 mg / L, the GABA content in the supernatant of Pediococcus acidilactici LF239 was 1131.42 mg / L, and the GABA content in the supernatant of Pediococcus acidilactici LH349 was 1164.29 mg / L. Thus, the GABA content of Pediococcus acidilactici TCI550 is nearly 2000 mg / L, which is much higher than that of commercially available GABA products.

[0074] Example 3: Analysis of the GABA content secreted by Pediococcus acidilactici TCI550-HPLC detection

[0075] First, the Pediococcus acidilactici TCI550 identified in Example 1 was inoculated into a liquid medium and cultured at 37°C in an anaerobic environment (i.e., the oxygen concentration in the culture environment was less than 1 vol%) for 24 hours to obtain a TCI550 bacterial solution. At this time, the liquid medium included 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of monopotassium phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0076] Next, the TCI550 bacterial solution cultured for 24 hours was centrifuged at 10,000 x g for 10 minutes to obtain a TCI550 supernatant, and then the TCI550 supernatant was filtered through a 0.22-um filter to obtain a TCI550 filtrate, which was used for subsequent high performance liquid chromatography (HPLC) to detect the GABA content. At this time, the HPLC analysis method was based on the research of Somasundaram et al. (Somasundaram, S., Tran, K. N. T., Ravikumar, S., & Hong, S. H. (2017). Biochemical Engineering Journal, 120, 1-6.).

[0077] 0.2 mL of the TCI550 filtrate was mixed with 0.1 mL of a 0.5 mol / L sodium bicarbonate (NaHCO3, purchased from Sigma-Aldrich, USA) solution, and then 0.02 mL of 1% (v / v) FDBN (purchased from Sigma-Aldrich, USA; diluted with acetonitrile) and 0.18 mL of ultrapure water were added and shaken well. Next, the reaction was performed in a 60°C water bath for 1 hour in the dark to obtain a reaction solution. The reaction solution was cooled to room temperature, 0.4 mL of a 0.01 mol / L potassium dihydrogen phosphate (KH2PO4, purchased from Sigma-Aldrich, USA) solution was added, and the mixture was shaken well, allowed to stand for 15 minutes, and then centrifuged to obtain a test solution.

[0078] 10 μL of the test solution was taken for analysis on an HPLC instrument. The HPLC instrument used an Agilent TC-C18 column (4.6 mm x 250 mm; purchased from Agilent Technologies, Taiwan). The mobile phase A (A solution) was 0.05 mol / L sodium acetate buffer solution (pH 5.7, adjusted to pH with 2% glacial acetic acid, and added 30 mL / L tetrahydrofuran), and the mobile phase B (B solution) was methanol (purchased from Sigma-Aldrich, USA). The flow rate used was 1 mL / min. The column temperature used was 28°C. The detection wavelength used was 360 nm.

[0079] The elution procedure used is shown in Table 2.

[0080] Table 2

[0081] Analysis results: The GABA concentration in the supernatant of TCI550 cultured for 24 hours was 2131.10 ppm.

[0082] Example 4: Detection of the content of γ-aminobutyric acid (GABA) secreted by nerve cells promoted by Pediococcus acidilactici TCI550

[0083] The cell culture medium used was DMEM medium (Dulbecco's modified Eagle's medium; purchased from Gibco, Cat. No. 11965-092) added with 10% fetal bovine serum (FBS; purchased from Gibco, Cat. No. 10437-028) and 1% Anti-Anti (purchased from Gibco).

[0084] The nerve cells used were human nerve cell SHSY-5Y cell strain (ATCC® CRL-2266 TM ; hereinafter referred to as SHSY-5Y cells).

[0085] The detection kit used was: GABA detection kit (brand CLOUD-CLONE CORP; product number CEA900Ge ELISA Kit for Gamma-Aminobutyric Acid (GABA)).

[0086] The sample to be tested used was TCI550 filtrate prepared from Pediococcus acidilactici TCI550 identified in Example 1. Pediococcus acidilactici TCI550 was inoculated into a liquid medium and cultured at 37°C in an anaerobic environment (i.e., an oxygen concentration of less than 1% by volume in the culture environment) for 24 hours to obtain TCI550 broth. Then, the TCI550 broth cultured for 24 hours was centrifuged at 10,000 x g for 10 minutes to obtain TCI550 supernatant, and the TCI550 supernatant was filtered through a 0.22-μm filter to obtain TCI550 filtrate. At this time, the liquid medium included 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of monopotassium phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate 80, 0.02 g / L of antifoam 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0087] 1 x 105 5 SHSY-5Y cells were inoculated into a 6-well culture plate containing 2 mL of cell culture medium and cultured at 37°C overnight.

[0088] Then, the cell culture medium of the culture plate inoculated with SHSY-5Y cells was replaced with experimental medium and cultured at 37°C for 6 hours. The experimental medium was different depending on the group, and the SHSY-5Y cells were divided into a control group and an experimental group. The experimental medium of the control group was cell culture medium, and the experimental medium of the experimental group was cell culture medium containing 0.25% of TCI550 filtrate.

[0089] Then, the SHSY-5Y cells cultured in each group were collected and treated with cell lysis buffer, and then centrifuged at 12,000 x g at 4°C for 10 minutes to remove cell debris and other insoluble substances of the SHSY-5Y cells. The supernatant after centrifugation was the sample to be tested.

[0090] The samples to be tested of the experimental group and the control group and the cell culture medium not cultured with SHSY-5Y cells (as a blank group) were detected for GABA content using a GABA detection kit and an ELISA reader. The value detected in the blank group was considered to be 100%, and was used as a basis for comparison to calculate the relative percentage of the experimental group and the control group. The experimental results are shown in FIG. 2 (*P<0.05; **P<0.01). The wavelength of the ELISA reader was set to 450 nm.

[0091] Please refer to Figure 2. The GABA content of the blank group was 100%, and the GABA content of the control group was 102.90%, and there was no significant difference between the two, representing no difference in GABA content. The GABA content of the experimental group was 109.02%, which was significantly different from the blank group and the control group, representing that the metabolic product of Lactococcus lactis TCI550 (i.e. TCI550 filtrate) can promote SHSY-5Y cells to secrete GABA.

[0092] Example 5: Brain-derived neurotrophic factor (BDNF) gene expression analysis

[0093] Here, the detected gene is the brain-derived neurotrophic factor (BDNF) gene (GeneID: 627) (hereinafter referred to as the BDNF gene). The BDNF gene is related to neural plasticity, anti-depression, and cognitive enhancement.

[0094] The cell culture medium used is DMEM medium (Dulbecco's modified Eagle's medium; purchased from Gibco, Cat. No. 11965-092) added with 10% fetal bovine serum (Fetal bovine serum FBS; purchased from Gibco, Cat. No. 10437-028) and 1% antibiotic (Anti-Anti; purchased from Gibco).

[0095] The neural cells used are human neural cells SHSY-5Y cell strain (ATCC® CRL-2266 TM ; hereinafter referred to as SHSY-5Y cells).

[0096] The sample to be tested used was TCI550 filtrate prepared from Pediococcus acidilactici TCI550 identified in Example 1. Pediococcus acidilactici TCI550 was inoculated into a liquid medium and incubated at 37°C in an anaerobic environment (i.e. an oxygen concentration of less than 1% by volume in the incubation environment) for 24 hours to obtain TCI550 bacterial solution. Next, the TCI550 bacterial solution incubated for 24 hours was centrifuged at 10,000 x g for 10 minutes to obtain TCI550 supernatant, and the TCI550 supernatant was filtered with a 0.22-um filter to obtain TCI550 filtrate. At this point, the liquid medium included 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of monopotassium phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0097] 1 x 105SHSY-5Y cells were inoculated into 2 mL of cell culture medium and incubated at 37°C overnight. 5 1 x 105SHSY-5Y cells were inoculated into 2 mL of cell culture medium and incubated at 37°C overnight.

[0098] Next, the cells were divided into three groups, namely a blank group, a control group, and an experimental group. The blank group was simply the cell culture medium (without cultured cells), and the aforementioned incubated SHSY-5Y cells were divided into the control group and the experimental group. The cell culture medium of the two groups was replaced with experimental culture medium and incubated at 37°C for 6 hours. At this point, the experimental culture medium of the control group was the cell culture medium, and the experimental culture medium of the experimental group was the cell culture medium containing 0.25% of the TCI550 filtrate.

[0099] The SHSY-5Y cells of each group were collected, and the RNA of each group was extracted with an RNA extraction reagent kit (purchased from Geneaid Corporation, Taiwan, Lot No. FC24015-G). Next, 2000 nanograms (ng) of RNA of each group was used as a template to synthesize cDNA by reverse transcription using a reverse transcription kit (purchased from Geneaid Corporation, Taiwan, Lot No. FC24015-G). III Reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) was used to reverse transcribe RNA into corresponding cDNA. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on the cDNA of each group by using ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA), KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000), and primers of Table 3 (SEQ ID NO: 9 to SEQ ID NO: 10) to observe the expression level of BDNF gene in SHSY-5Y cells. The instrument setting condition of qRT-PCR was 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated for 40 cycles, and the relative quantification of genes was performed by using SCORE method, as shown in Figure 3. Here, qRT-PCR performed on cDNA can indirectly quantify the mRNA expression level of the gene, and thus infer the expression level of the protein encoded by the gene. Moreover, the gene expression level of BDNF in Figure 3 is presented in relative fold, wherein the expression level of the blank group is considered as 1, and serves as the basis for calculating the gene expression level of the control group and the experimental group.

[0100] Table 3

[0101] In Table 3, F is forward primer, and R is reverse primer.

[0102] Referring to Figure 3, the expression level of BDNF gene in the blank group is 1.00. Compared with the blank group, the expression level of BDNF gene in the control group is 82.55, and the expression level of BDNF gene in the experimental group is 363.23. Therefore, the expression level of BDNF gene in the experimental group is significantly improved, which is about 360 times the expression level of the blank group and 4 times the expression level of the control group, indicating that the metabolic product of Pediococcus acidilactici TCI550 (i.e., TCI550 filtrate) can effectively promote the expression of BDNF gene in nerve cells and increase the expression level of BDNF. In other words, when the subject takes Pediococcus acidilactici TCI550, the expression level of BDNF gene can be increased, thereby helping to improve neural plasticity and brain strength.

[0103] Example 6: Expression analysis of serotonin production-related genes, melatonin synthesis-related genes, and anti-depression-related genes

[0104] Here, the serotonin production-related genes tested were the tryptophan hydroxylase 1 (TPH1) gene (Gene ID: 7166) (hereinafter referred to as the TPH1 gene) and the dopamine decarboxylase (DDC) gene (Gene ID: 1644) (hereinafter referred to as the DDC gene). The melatonin synthesis-related genes tested were the acetylserotonin O-methyltransferase (ASMT) gene (Gene ID: 438) (hereinafter referred to as the ASMT gene) and the arylalkylamine acetyltransferase (AANAT) gene (Gene ID: 15) (hereinafter referred to as the AANAT gene). The antidepressant-related gene tested was the sirtuin 1 (SIRT1) gene (Gene ID: 23411) (hereinafter referred to as the SIRT1 gene).

[0105] The cell culture medium used was DMEM (Dulbecco's modified Eagle's medium; purchased from Gibco, Cat. No. 11965-092) supplemented with 10% fetal bovine serum (FBS; purchased from Gibco, Cat. No. 10437-028) and 1% antibiotic (Anti-Anti; purchased from Gibco).

[0106] The neural cells used were human neural cell line SHSY-5Y ( CRL-2266 TM ; hereinafter referred to as SHSY-5Y cells).

[0107] The test sample used was a TCI550 filtrate prepared from the Pediococcus acidilactici TCI550 identified in Example 1. Pediococcus acidilactici TCI550 was inoculated into a liquid culture medium and cultured at 37°C in an anaerobic environment (i.e., an oxygen concentration of less than 1% by volume) for 24 hours to obtain a TCI550 bacterial liquid. The 24-hour cultured TCI550 bacterial liquid was then centrifuged at 10,000 x g for 10 minutes to obtain a TCI550 supernatant. The TCI550 supernatant was then filtered through a 0.22 μm filter membrane to obtain a TCI550 filtrate. Here, the liquid culture medium contains 25g / L yeast peptone 902, 10g / L yeast peptone GLSF, 6g / L dipotassium hydrogen phosphate, 3g / L potassium dihydrogen phosphate, 1g / L cysteine, 5g / L sodium acetate, 0.1g / L magnesium sulfate, 1g / L polysorbate 80, 0.02g / L defoamer 30PS, 50g / L glucose, 20g / L 10N sodium hydroxide, 0.16g / L manganese gluconate, 1.7g / L citric acid, and 15g / L soy peptide powder.

[0108] Take 1×10 5 SHSY-5Y cells were inoculated into 2 mL of cell culture medium and cultured at 37°C overnight.

[0109] The cells were then divided into three groups: a blank group, a control group, and an experimental group. The blank group contained pure cell culture medium (no cultured cells). The SHSY-5Y cells were then divided into a control group and an experimental group. The cell culture medium in both groups was replaced with the experimental medium, and the cells were incubated at 37°C for 6 hours. The experimental medium in the control group consisted of cell culture medium, while the experimental group contained cell culture medium supplemented with 0.25% TCI550 filtrate.

[0110] SHSY-5Y cells from each group were collected and RNA was extracted using an RNA extraction kit (purchased from Geneaid, Taiwan, China, Lot No. FC24015-G). III Reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) was used to reverse transcribe RNA into corresponding cDNA. Quantitative real-time reverse transcription polymerase chain reaction was performed on the cDNA of each group by using ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA), KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000), and primers of Table 4 (SEQ ID NO: 11 to SEQ ID NO: 20) to observe the expression levels of various genes in SHSY-5Y cells. The instrument setting conditions of quantitative real-time reverse transcription polymerase chain reaction were 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated for 40 cycles, and the relative quantification of genes was performed using the SCORE method, as shown in FIG. 4. Here, quantitative real-time reverse transcription polymerase chain reaction by cDNA can indirectly quantify the mRNA expression level of the gene, and thus infer the expression level of the protein encoded by the gene. Moreover, the expression levels of genes such as serotonin production-related genes, melatonin synthesis-related genes, and anti-depression-related genes in FIG. 4 are presented as relative percentages, with the expression level of the blank group being considered as 100%, and serving as the basis for calculating the expression levels of genes in the control group and the experimental group.

[0111] Table 4

[0112] In Table 4, F is the forward primer, and R is the reverse primer.

[0113] Referring to FIG. 4, the expression levels of the TPH1 gene, the DDC gene, the ASMT gene, the AANAT gene, and the SIRT1 gene of the blank group were all 100%. Compared with the blank group, the expression level of the TPH1 gene of the control group was 118.35%, the expression level of the DDC gene was 100.23%, the expression level of the ASMT gene was 100.68%, the expression level of the AANAT gene was 101.37%, and the expression level of the SIRT1 gene was 100.23%, representing that the SHSY-5Y cells were not affected by the pure cell culture medium. The expression level of the TPH1 gene of the experimental group was 138.79%, the expression level of the DDC gene was 106.97%, the expression level of the ASMT gene was 110.56%, the expression level of the AANAT gene was 106.25%, and the expression level of the SIRT1 gene was 108.62%. Therefore, the expression amounts of the genes of the experimental group were significantly improved, representing that the metabolic products of the Pediococcus acidilactici TCI550 (i.e., the TCI550 filtrate) can effectively promote the expression of the serotonin production-related genes (such as the TPH1 gene and the DDC gene), the melatonin synthesis-related genes (such as the ASMT gene and the AANAT gene), and the anti-depression-related genes (such as the SIRT1 gene), and increase the expression amounts of the corresponding proteins of the genes. In other words, when the subject takes the Pediococcus acidilactici TCI550, the expression amounts of the TPH1 gene, the DDC gene, the ASMT gene, the AANAT gene, and the SIRT1 gene can be increased, thereby helping to protect the nerves, improve the cognition, and resist the depression, and the like.

[0114] Example 7: Human test

[0115] Test method: The live bacteria capsules prepared from the Pediococcus acidilactici TCI550 identified in Example 1 (hereinafter referred to as the Pediococcus acidilactici TCI550 live bacteria capsules) were tested, and the Pediococcus acidilactici TCI550 live bacteria capsules contained 50 mg of bacterial powder. Ten subjects took one Pediococcus acidilactici TCI550 live bacteria capsule after dinner / sleep every day for four consecutive weeks, and blood, sleep tests, and somatic questionnaire surveys were conducted before taking (week 0), after taking for two weeks (week 2), and after taking for four weeks (week 4). Among them, the ten subjects were over 20 years old and self-conscious of poor sleep quality (for example, difficulty falling asleep, easy to wake up, or early wake up). Among them, the bacterial powder was prepared by adding the Pediococcus acidilactici TCI550 bacterial liquid prepared in Example 2 (i.e., the bacterial liquid to be tested in Example 2) to 1.25% (w / w) soy milk powder, 1.25% (w / w) trehalose, 0.5% (w / w) indigestible malt dextrin, and 0.3% (w / w) sorbitol, mixing them uniformly, and freeze-drying and grinding.

[0116] Test items: Blood test - GABA and BDNF content detection in blood; Autonomic nervous system test - sympathetic nerve cell activity (LF) detection; Somatosensory questionnaire - sleep disorders and cognitive dysfunction.

[0117] 7-1. Blood test

[0118] Blood test method: 10 subjects were tested before taking (i.e. week 0), after taking for 2 weeks (week 2), and after taking for 4 weeks (i.e. week 4), blood samples were collected and entrusted to Dajiang Gene Medicine Co., Ltd. to detect the content of gamma-aminobutyric acid (GABA) (as shown in Figure 5) and brain-derived neurotrophic factor (BDNF) (as shown in Figure 6) in the blood of the subjects.

[0119] Please refer to Figure 5. Before taking (i.e. week 0), the average GABA content in the blood of 10 subjects was 100.58 ng / mL. After continuous taking for 2 weeks (i.e. week 2), the average GABA content in the blood of 10 subjects was 202.32 ng / mL, which was 101.1% higher than that of week 0. After continuous taking for 4 weeks (i.e. week 4), the average GABA content in the blood of 10 subjects was 356.40 ng / mL, which was 254.3% higher than that of week 0. In other words, continuous taking of Pediococcus acidilactici TCI550 live bacteria capsules for 4 weeks can increase the content of GABA in the body. And the number of improved subjects among the 10 subjects reached 90% (i.e. 9 out of 10 subjects had significantly increased GABA content in their blood), more than half. Therefore, the receptor can increase the content of GABA in the body by taking Pediococcus acidilactici TCI550, so as to achieve the effects of calming and stabilizing the nerves, protecting the nerves, improving cognitive ability, etc.

[0120] Please refer to Figure 6. Before taking (i.e. week 0), the average BDNF content in the blood of 10 subjects was 4.93 ng / mL. After continuous taking for 2 weeks (i.e. week 2), the average BDNF content in the blood of 10 subjects was 5.66 ng / mL, which was 14.8% higher than that of week 0. After continuous taking for 4 weeks (i.e. week 4), the average BDNF content in the blood of 10 subjects was 6.41 ng / mL, which was 30% higher than that of week 0. In other words, continuous taking of Pediococcus acidilactici TCI550 live bacteria capsules for 4 weeks can increase the content of BDNF in the body. And the number of improved subjects among the 10 subjects reached 80% (i.e. 8 out of 10 subjects had significantly increased BDNF content in their blood), more than half. Therefore, the receptor can increase the content of BDNF in the body by taking Pediococcus acidilactici TCI550, so as to achieve the effects of calming and stabilizing the nerves, protecting the nerves, improving cognitive ability, improving sleep, improving brain plasticity, etc.

[0121] 7-2. Autonomic nervous system detection

[0122] When the autonomic nervous system is out of balance, the sympathetic nervous system is in an excited state during sleep, making the body and brain unable to rest properly. Therefore, by measuring the autonomic nervous activity in the sleep stage, it can be determined whether the autonomic nervous system is healthy or out of balance.

[0123] Detection method: 10 subjects used the LARGAN sleep detection system (LARGAN Cloud) at home to detect their heart rate variability (HRV) data during the 10 minutes of sitting before sleep. Heart rate variability (HRV) is an important indicator of autonomic nervous activity measurement. Among them, LF is the overall activity of the sympathetic nervous system, representing the degree of tension and excitement of the subjects.

[0124] In addition, the value of heart rate variability decreases with age, and the effect of gender on heart rate variability is less than age, and the gender difference decreases when the age is greater than 30 years old, so the age of 30 years old is used as the cut-off point for subsequent analysis. Among the 10 subjects, there were 4 subjects aged 20 to 30 years old, and 6 subjects aged 30 years old or older. The average age of the 4 subjects aged 20 to 30 years old was 26 years old; the average age of the 6 subjects aged 30 years old or older was 43 years old. The analysis results are shown in Figure 7.

[0125] Judgment criteria - LF reference value: 263.9 ms for age 20-30 years old 2 to 896.7 ms 2 ; 112.9 ms for age greater than 30 years old 2 to 543.2 ms 2 .

[0126] Please refer to Figure 7. The average sympathetic nervous cell activity of the 4 subjects aged 20 to 30 years old at week 0 was 962.7 ms 2 , which is higher than the general LF reference value, so the 4 subjects have the possibility of anxiety and insomnia. After continuous use for 4 weeks (i.e. week 4), the average sympathetic nervous cell activity of the 4 subjects aged 20 to 30 years old was 614.7 ms 2 , which is 36.1% lower than at week 0, representing the nervous state of the subjects from tension and excitement to relaxation and sleep. The average sympathetic nervous cell activity of the 6 subjects aged 30 years old or older at week 0 was 580.9 ms 2 , which is higher than the general LF reference value, so the 6 subjects have the possibility of anxiety and insomnia. After continuous use for 4 weeks (i.e. week 4), the average sympathetic nervous cell activity of the 6 subjects aged 30 years old or older was 388.8 ms2 , compared to a 3.1% decrease at week 0, representing the subject's nervous state from tension to relaxation. Thus, the receptor can alleviate the pre-sleep nerves by taking lactobacillus TCi550, thereby calming and stabilizing the nerves, maintaining the nerves, and helping sleep, etc., and when the sleep is sufficient, it can achieve various effects such as improving cognitive ability, improving brain plasticity, etc.

[0127] 7-3. Sleep disorder evaluation: Pittsburgh sleep questionnaire

[0128] Detection method: Pittsburgh sleep quality index (PSQI): Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) is used to evaluate the scores of seven aspects of subjective sleep quality, sleep latency, total sleep time, sleep efficiency, sleep disorders, sleep medication use, daytime dysfunction, etc. Each aspect has a minimum score of 0 and a maximum score of 3. The total score ranges from 0 to 21. When the PSQI score is greater than 5, it indicates that there is a sleep quality disorder, and the higher the score, the worse the sleep quality. Ten subjects filled out the Pittsburgh sleep quality index at weeks 0 and 4 to evaluate the sleep quality before and after taking lactobacillus TCi550. The evaluation results are shown in Figures 8 and 9.

[0129] Please refer to Figure 8. At week 0, the overall sleep disorder degree (PSQI) of the 10 subjects was 9, indicating that the subjects had sleep disorder problems. After continuous administration for 4 weeks (i.e. week 4), the overall sleep disorder degree (PSQI) of the 10 subjects decreased to 6, although there were still sleep quality disorders among the subjects, but the score also represented an increase in sleep quality. The number of subjects who felt improved was 80% (i.e. 8 subjects).

[0130] In addition to the overall sleep disorder degree score, the 10 subjects also evaluated the sleep disorder severity judgment items: "poor sleep quality, difficulty falling asleep, low total sleep time, and inability to be energetic and do things during the day". Week 0 scores were considered 100%, and the relative percentage of week 4 scores was converted to estimate the improvement.

[0131] Referring to FIG. 9, the severity of "poor sleep quality" decreased by 61.1%, the severity of "difficulty falling asleep" decreased by 43.8%, the severity of "low total sleep time" decreased by 64.3%, and the severity of "inability to be energetic and do things during the day" decreased by 69.6%. Among them, the scores of "poor sleep quality" and "difficulty falling asleep" decreased significantly by 38.9% and 56.2%, respectively, and the rate of the number of subjects who felt improved was 70% (i.e., 7 subjects) for both.

[0132] Therefore, it was found that the subjects could improve sleep quality and sleep disorder problems by taking Lactococcus lactis TCI550, and by improving sleep quality, the subjects could maintain neurological health and achieve the effect of neuroprotection.

[0133] 7-4. Cognitive impairment evaluation:

[0134] Evaluation method: The Taiwan Cognitive Function Scale is a diagnostic tool for evaluating whether a subject has memory deterioration, is prone to hesitation, and is unable to concentrate. The Taiwan Cognitive Function Scale can evaluate the degree of influence of cognitive function on daily life, and a total of 5 questions, with a higher score indicating a worse cognitive function status. The Taiwan Cognitive Function Scale was filled out by the 10 subjects at weeks 0 and 4 to evaluate the cognitive function before and after taking Lactococcus lactis TCI550. The evaluation results are shown in FIGS. 10 and 11.

[0135] Referring to FIG. 10, the cognitive impairment score of the 10 subjects was 5.3 at week 0. After 4 weeks of continuous administration (i.e., at week 4), the cognitive impairment score of the 10 subjects decreased to 1.7, representing a significant decrease of 67.9% in the cognitive impairment score of the 10 subjects. Moreover, the rate of the number of subjects who felt improved was 90% (i.e., 9 subjects).

[0136] In addition to the cognitive impairment score, the 10 subjects were also evaluated for the judgment item of cognitive impairment severity: "memory deterioration and decreased concentration". The scores at week 0 were all taken as 100%, and the relative percentages of the scores at week 4 were converted to infer the degree of improvement.

[0137] Referring to FIG. 11, the severity of "memory deterioration" decreased by 15.4% at week 4, and the severity of "decreased concentration" decreased by 42.9%. Among them, the 10 subjects felt that their memory and concentration were improved, and the severity decreased by 84.6% and 57.1%, respectively. Moreover, the rate of the number of subjects who felt that their memory was improved was 80% (i.e., 8 subjects), and the rate of the number of subjects who felt that their concentration was improved was 60% (i.e., 6 subjects).

[0138] Therefore, the subject can improve memory and concentration and improve cognitive function by taking Pediococcus acidilactici TCI550.

[0139] 7-5. Somatosensory questionnaire:

[0140] Detection method: 10 subjects filled out the somatosensory questionnaire after taking Pediococcus acidilactici TCI550 live bacterial capsules for 4 weeks, and at the 4th week. Among them, for the two questions of whether Pediococcus acidilactici TCI550 bacterial capsules "improve the frequency of waking up at night" and "improve concentration", three options of "no feeling", "slightly feeling" and "very feeling" are provided for selection. The questionnaire results are shown in Figures 12 and 13.

[0141] Please refer to Figure 12. For the effect of Pediococcus acidilactici TCI550 bacteria on "improving the frequency of waking up at night", 50% of the 10 subjects felt "very feeling", 30% of the subjects felt "slightly feeling", and 20% of the subjects felt "no feeling". Therefore, 8 subjects felt that Pediococcus acidilactici TCI550 bacteria improved the frequency of waking up at night.

[0142] Please refer to Figure 13. For the effect of Pediococcus acidilactici TCI550 bacteria on "improving concentration", 20% of the 10 subjects felt "very feeling", 50% of the subjects felt "slightly feeling", and 30% of the subjects felt "no feeling". Therefore, 7 subjects felt that Pediococcus acidilactici TCI550 bacteria improved concentration.

[0143] Therefore, long-term use of Pediococcus acidilactici TCI550 can effectively improve the sleep quality of the subject and improve their concentration.

[0144] Of course, the present application can have other various embodiments, and those skilled in the art can make various corresponding changes and modifications according to the present application without departing from the spirit and essence of the present application. However, these corresponding changes and modifications should all belong to the protection scope of the claims of the present application.

[0145] Industrial applicability

[0146] In summary, the Pediococcus acidilactici TCI550 with the accession number of DSM34536 according to any embodiment of the present application can be used to prepare a neuro-health composition, a cognition-enhancing composition, or a combination thereof. In other words, the aforementioned composition has one or more of the following functions: improving brain plasticity, calming nerves, reducing overall activity of sympathetic nerve cells, improving reaction force, concentration, memory, improving reaction force, concentration, memory, enhancing nerve cell serotonin production-related genes (e.g., TPH1 gene, DDC gene, or a combination thereof), melatonin synthesis-related genes (e.g., ASMT gene, AANAT gene, or a combination thereof), anti-depression-related genes (e.g., SIRT1 gene, BDNF gene, or a combination thereof), enhancing γ-aminobutyric acid content in blood, enhancing brain-derived neurotrophic factor content in blood, and the like.

Claims

1. Use of Pediococcus acidilactici and / or its metabolite for the preparation of a neurohealth composition, wherein the Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the accession number DSM34536.

2. The use of claim 1, wherein the neurohealth is improving brain plasticity, calming the nerves, or a combination thereof.

3. The use of claim 1, wherein the composition has the ability to reduce the overall activity of sympathetic nerve cells.

4. The use of claim 1, wherein the composition has the ability to elevate the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in nerve cells.

5. The use of claim 4, wherein the serotonin production-related gene is a Tryptophan hydroxylase (TPH) gene, a Dopa decarboxylase (DDC) gene, or a combination thereof.

6. The use of claim 4, wherein the melatonin synthesis-related gene is an Acetylserotonin O-methyltransferase (ASMT) gene, an Arylalkylamine N-acetyltransferase (AANAT) gene, or a combination thereof.

7. The use of claim 4, wherein the anti-depression-related gene is a Sirtuin 1 (SIRT1) gene, a Brain-Derived Neurotrophic Factor (BDNF) gene, or a combination thereof.

8. The use of claim 1, wherein the composition has the ability to promote nerve cells to secrete γ-aminobutyric acid (GABA).

9. The use of claim 1, wherein the Pediococcus acidilactici TCI550 has the ability to produce γ-aminobutyric acid and elevate the content of the γ-aminobutyric acid in blood.

10. The use of claim 1, wherein the Pediococcus acidilactici TCI550 has the ability to elevate the content of Brain-Derived Neurotrophic Factor (BDNF) in blood.

11. The use of claim 1, wherein the Pediococcus acidilactici TCI550 is used in an amount of 50 mg per day.

12. Use of Pediococcus acidilactici and / or its metabolite for the preparation of a cognitive enhancement composition, wherein the Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the accession number DSM34536.

13. The use of claim 12, wherein the composition has the ability to improve reactivity, focus, memory, or a combination thereof.

14. The use of claim 12, wherein the composition has the ability to elevate the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in nerve cells.

15. The use of claim 14, wherein the serotonin production-related gene is a Tryptophan hydroxylase (TPH) gene, a Dopa decarboxylase (DDC) gene, or a combination thereof.

16. The use of claim 14, wherein the melatonin synthesis-related gene is an Acetylserotonin O-methyltransferase (ASMT) gene, an Arylalkylamine acetyltransferase (AANAT) gene, or a combination thereof.

17. The use of claim 14, wherein the anti-depression-related gene is a Sirtuin 1 (SIRT1) gene, a Brain-Derived Neurotrophic Factor (BDNF) gene, or a combination thereof.

18. The use of claim 12, wherein the composition has the ability to promote the secretion of γ-aminobutyric acid (GABA) by nerve cells.

19. The use of claim 12, wherein the Pediococcus acidilactici TCI550 has the ability to produce and elevate the content of γ-aminobutyric acid (GABA) in blood.

20. The use of claim 12, wherein the Pediococcus acidilactici TCI550 has the ability to elevate the content of Brain-Derived Neurotrophic Factor (BDNF) in blood.

21. The use of claim 12, wherein the Pediococcus acidilactici TCI550 is used in an amount of 50 mg per day.

22. A Pediococcus acidilactici, which is Pediococcus acidilactici TCI550 having the accession number DSM34536.

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